The ten amino acids of the oxygen-evolving enhancer of tobacco is sufficient as the peptide residues for protein transport to the chloroplast thylakoid.

KEY MESSAGE: The thylakoid transit peptide of tobacco oxygen-evolving enhancer protein contains a minimal ten amino acid sequences for thylakoid lumen transports. This ten amino acids do not contain twin-arginine, which is required for typical chloroplast lumen translocation. Chloroplasts are intracellular organelles responsible for photosynthesis to produce organic carbon for all organisms. Numerous proteins must be transported from the cytosol to chloroplasts to support photosynthesis. This transport is facilitated by chloroplast transit peptides (TPs). Four chloroplast thylakoid lumen TPs were isolated from Nicotiana tabacum and were functionally analyzed as thylakoid lumen TPs. Typical chloroplast stroma-transit peptides and thylakoid lumen transit peptides (tTPs) are found in N. tabacum transit peptides (NtTPs) and the functions of these peptides are confirmed with TP-GFP fusion proteins under fluorescence microscopy and chloroplast fractionation, followed by Western blot analysis. During the functional analysis of tTPs, we uncovered the minimum 10 amino acid sequence is sufficient for thylakoid lumen transport. These ten amino acids can efficiently translocate GFP protein, even if they do not contain the twin-arginine residues required for the twin-arginine translocation (Tat) pathway, which is a typical thylakoid lumen transport. Further, thylakoid lumen transporting processes through the Tat pathway was examined by analyzing tTP sequence functions and we demonstrate that the importance of hydrophobic core for the tTP cleavage and target protein translocation.

Metabolism of phenolic compounds catalyzed by Tomato CYP736A61.

Cytochrome P450s (CYPs) regulate plant growth and stress responses by producing diverse primary and secondary metabolites. However, the function of many plant CYPs remains unknown because, despite their structural similarity, predicting the enzymatic activity of CYPs is difficult. In this study, one member of the CYP736A subfamily (CYP736A61) from tomatoes was isolated and characterized its enzymatic functions. CYP736A61 was successfully expressed in Escherichia coli through co-expression with molecular chaperones. The purified CYP736A61 showed hydroxylation activity toward 7-ethoxycoumarin, producing 7-hydroxycoumarin or 3-hydroxy 7-ethoxycoumarin. Further substrate screening revealed that dihydrochalcone and stilbene derivates (resveratrol and polydatin) are the substrates of CYP736A61. CYP736A61 also mediated the hydroxylation of resveratrol and polydatin, albeit with low activity. Importantly, CYP736A61 mediated the cleavage of resveratrol and polydatin as well as pinostilbene and pterostilbene. Interestingly, CY736A61 also converted phloretin to naringenin chalcone. These results suggest that CYP736A61 is a novel CYP enzyme with stilbene cleavage activity.

Development of a Genome-Edited Tomato With High Ascorbate Content During Later Stage of Fruit Ripening Through Mutation of SlAPX4.

Ascorbate is an essential antioxidant substance for humans. Due to the lack of ascorbate biosynthetic enzyme, a human must intake ascorbate from the food source. Tomato is one of the most widely consumed fruits, thus elevation of ascorbate content in tomato fruits will improve their nutritional value. Here we characterized Solanum lycopersicum ASCORBATE PEROXIDASE 4 (SlAPX4) as a gene specifically induced during fruit ripening. In tomatoes, ascorbate accumulates in the yellow stage of fruits, then decreases during later stages of fruit ripening. To investigate whether SlAPX is involved in the decrease of ascorbate, the expression of SlAPXs was analyzed during fruit maturation. Among nine SlAPXs, SlAPX4 is the only gene whose expression was induced during fruit ripening. Mutation of SlAPX4 by the CRISPR/Cas9 system increased ascorbate content in ripened tomato fruits, while ascorbate content in leaves was not significantly changed by mutation of SlAPX4. Phenotype analysis revealed that mutation of SlAPX4 did not induce an adverse effect on the growth of tomato plants. Collectively, we suggest that SlAPX4 mediates a decrease of ascorbate content during the later stage of fruit ripening, and mutation of SlAPX4 can be used for the development of genome-edited tomatoes with elevated ascorbate content in fruits.